Heterogenous specific binding assay employing a cycling reactant as label

ABSTRACT

An improved heterogenous specific binding assay method which employs a substance having reactant activity, i.e., a reactant, as a labeling substance in the detection of a ligand in a liquid medium. The method is carried out using reagent means which comprises, as its labeled constituent, a conjugate formed of a specific binding substance coupled to the reactant. The reactant advantageously is an enzymatic reactant such as an enzyme substrate or coenzyme. The activity of the conjugated reactant as a constituent of a predetermined reaction system is utilized as means for monitoring the extent of binding of the labeled constituent in conventional heterogenous specific binding assay schemes. The presence of a ligand in a liquid medium may be determined following conventional competitive binding manipulative techniques. After the necessary separation of the bound-and free-phases resulting in the specific binding reaction system, the extent of binding of the labeled constituent is determined by contacting either phase with the necessary materials to form the predetermined monitoring reaction system in which the labeling substance is active and assessing reactant activity therein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a division of application Ser. No. 894,838, filed Apr. 10, 1978,U.S. Pat. No. 4,230,797, which is a continuation of application Ser. No.667,982, filed Mar. 18, 1976, now abandoned, which is acontinuation-in-part of application Ser. No. 572,008, filed Apr. 28,1975, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods and means for determining the presenceof a ligand in a liquid medium based on the affinity of the ligand for aspecific binding partner thereof. In particular, this invention relatesto methods and means for use in specific binding assays which do notemploy radioactive materials or modified enzymes as the labelingsubstance.

The desirability of a convenient, reliable, and nonhazardous means fordetecting the presence of low concentrations of substances in liquids isself-evident. This is particularly true in the field of clinicalchemistry where constituents of body fluids which may appear inconcentrations as low as 10⁻¹¹ molar are known to be of pathologicalsignificance. The difficulty of detecting such low concentrations iscompounded in the field of clinical chemistry where sample size isusually quite limited.

Classically, substances have been detected in liquids based on areaction scheme wherein the substance to be detected is a necessaryreactant. The presence of unknown is indicated by the appearance of areaction product or the disappearance of a known reactant. In certaininstances, such an assay method may be quantitative, based on ameasurement of either the rate of appearance of product or disappearanceof reactant or measurement of the aggregate amount of product producedor reactant consumed in attaining equilibrium. Each assay reactionsystem is necessarily either limited to use in the detection of only asmall group of substances or is non-specific.

2. Description of the Prior Art

The search for assay systems which are highly specific yet adaptable tothe detection of a wide range of substances has evolved theradioimmunoassay. In this system a known amount of a radiolabeled formof the substance to be detected is allowed to compete with the unknownfor a limited quantity of antibody specific for the unknown. The amountof the labeled form that becomes bound to antibody varies inversely withthe level of unknown present. Inherent in the radioimmunoassay techniqueis the need to separate the labeled form of substance to be detectedwhich becomes bound to antibody, the bound-phase, from that which doesnot become so bound, the free-phase. While various ways of accomplishingthe required separation have been developed, as exemplified in U.S. Pat.Nos. 3,505,019; 3,555,143; 3,646,346; 3,720,760; and 3,793,445, allrequire at least one separate manipulative step, such as filtering,centrifuging, washing, or draining a column to insure efficientseparation of the bound and free phases. Such separation is oftenaccomplished by forming a system comprised of an insoluble portioncontaining the bound-phase and a liquid portion containing thefree-phase such that the amount of radioactive label in either portionis a function of the extent of binding of the labeled material, and thusa function of the amount of ligand in the sample tested. The term"heterogeneous" as generally used by the scientific community and asapplied herein, means those specific binding assays wherein a separationof the bound- and free-phases is accomplished. Such a separation isnecessary to carry out a specific binding assay where the labeledmaterial in the bound-phase is indistinguishable from that in thefree-phase.

Because of the hazard and difficulty of handling radioactive materials,there have been many attempts to devise convenient specific bindingassay systems which are as sensitive and rapid as radioimmunoassays butwhich utilize features other than radioactivity as the means formonitoring the binding reaction. As will be discussed more fullyhereinafter, materials which have been utilized as the labelingsubstance in place of radioactive atoms or molecules include suchdiverse materials as enzymes, fluoroescent molecules, andbacteriophages.

Exemplary of methods which have been developed using an enzyme as thelabeling substance are those described in U.S. Pat. Nos. 3,654,090;3,791,932; 3,839,153; 3,850,752; and 3,879,262 and in the Journal ofImmunological Methods 1:247(1972) and the Journal of Immunology109:129(1972). In each of the described methods an enzyme is chemicallycoupled to either the ligand to be detected or a binding partner thereofand an appropriate heterogeneous specific binding reaction scheme isconstructed whereby after incubation with a sample, the amount ofenzymatic activity associated with either the insoluble portion or theliquid portion is a function of the amount of ligand in the sample. Theproblems associated with the synthesis and characterization of theenzyme-conjugates are serious short comings of this approach.

Of interest is the enzyme-tagged immunoassay described in U.S. Pat. No.3,817,837. This method does not require the use of a partitioned (i.einsoluble portion/liquid portion) specific binding reaction system andthe separation procedure necessitated thereby since the enzyme-taggedligand is designed such that upon reaction with the binding partner ofthe ligand, enzymatic activity is inhibited. Thus, the ratio of boundtagged material to that in free form can be determined by monitoringchanges in enzymatic activity. Nonetheless, ths method suffers from thedifficulty of preparing well-characterized enzyme-tagged conjugates andof finding enzymes that will fit the basic design of the system.

British Pat. No. 1,392,403 and French Pat. No. 2,201,299, which patentscorrespond to U.S. Pat. No. 3,880,934 describe a specific binding assaywhich utilizes a non-active precursor of a spectrophotometrically-activesubstance as the labeling substance. After incubation of the sample withthe specific binding reaction system, the insoluble and liquid portionsare separated and the amount of labeling substance present in the liquidportion, which is a function of the amount of ligand to be detected inthe sample, is determined by carrying out reaction steps that transformthe inactive labeling substance into a chromogen of fluorometricallyactive material which is then measured by conventional means.

Other specific binding assay methods employing different types oflabeling substances are disclosed in: U.S. Pat. No. 3,850,578 whichdiscloses the use of electron spin resonance as a labeling means; U.S.Pat. No. 3,901,654 which discloses the use of fluorescense quenching andenhancement as a labeling means; and Report No. PB-224,875 of theNational Technical Information Service (NTIS) of the U.S. Department ofCommerce (1973) which describes an unsuccessful attempt to use heminchloride as a labeling substance in a heterogeneous assay systemmonitored by a chemiluminescence reaction. Nature 219:186(1968)describes in great detail certain radioimmunoassay procedures and makesa passing reference of a very general nature to the possible use ofcoenzymes and viruses in place of radioisotopes as labeling substances.However, the author provides no enlightenment as to how to carry out anassay using such alternative labeling substances, or in fact as towhether such an assay would be operable. For further background,reference may be had to Principles of Competitive Protein-BindingAssays, ed. Odell and Daughaday (J. B. Lippincott Co., Philadelphia,1972) which discusses in breadth the various known assay schemes and thedifferent materials and features that hae been used as labels forspecific binding assays.

Even though many new types of specific binding assays have beensuggested and investigated, the radioimmunoassay and the variousenzyme-tagged immunoassays remain the most widely used and improved.However, both types of systems have obvious shortcomings, theradioimmunoassay in its use of radioactive material which is hazardousand requires careful handling and the enzyme-tagged immunoassays in thedifficulty of preparing useful enzyme-tagged conjugates.

It is therefore an object of the present invention to provide a novelmethod and means for detecting a ligand in a liquid which do not employinconvenient radioactive materials or modified enzymes as the labelingsubstance.

Further, it is an object of the present invention to provide aheterogeneous specific binding assay method and means which are moreversatile and convenient than those of the prior art.

Another object of the present invention is to provide a heterogeneousspecific binding assay method and means which employ a labelingsubstance which is capable of being coupled to the ligand or to aspecific binding partner thereof more conveniently than can the enzymeof the prior art method.

It is also an object of the present invention to provide a heterogeneousspecific binding assay method and means which employ a conjugatecomprising a labeling substance which is more conveniently detectableusing a wide variety of sensitive monitoring reaction systems than isthe enzyme in the prior art method.

SUMMARY OF THE INVENTION

The present invention provides a highly convenient, versatile, andsensitive improved heterogeneous specific binding assay method and meansbased on the use of, as labeling substance, a substance which exhibitsreactant activity as a constituent of a predetermined reaction system,such substance being referred to herein as the reactant. The inventivelabeling substance may be used in any of the conventional heterogeneousspecific binding assay schemes. The amount of the reactant present ineither of the bound- and free-phases is determined by contacting eitherphase with at least one reagent which forms, with the reactant, thepredetermined reaction system which serves as means for monitoring thespecific biding reaction. Quantitative determinations are carried out bycomparing the amount of reactant activity measured in one phase to thoseproduced by the same assay of liquid media containing known amounts ofthe ligand under determination.

The improved method generally comprises the steps of (a) contacting theliquid test medium with reagent means which includes a labeledconstituent comprising a conjugate of a reactant as defined herein, aslabeling substance, and a binding component and which forms, with theligand to be determined, a binding reaction system producing abound-phase and a free-phase of said labeled constituent, the quantityof said labeling substance resulting in said bound-phase being afunction of the amount of the ligand present in the test medium; (b)separating said bound-phase from said free-phase, and (c) determiningthe quantity of said reactant in said bound- or free-phase, and therebythe amount of the ligand in the test medium, by assessing the reactantactivity therein. As will be more fully discussed hereinafter, thebinding reaction system may take the form of any of the knownconventional techniques such as those employed in radioimmunoassaysystems and in heterogeneous enzyme immunoassay systems.

The monitoring reaction system preferably is enzyme-catalyzed. Usually,a monitoring reaction system is selected which is highly sensitive tothe reactant in the conjugate. Luminescent or fluorescent reactionsystems are very useful in this regard. Particularly preferred arecyclic reaction systems, especially those in which the reactant is thecycled material. Of the preferred cyclic reaction systems, those whichare enzyme-catalyzed are particularly advantageous. The reactant in theconjugate is usually an enzymatic reactant, such as an enzyme substrateor, as is particularly preferred, a coenzyme, and preferably has amolecular weight of less than 9000.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the context of this disclosure, the following terms shall be definedas follows: ligand is the substance, or group of substances, whosepresence or the amount thereof in a liquid medium is to be determined;specific binding partner of the ligand is any substance, or group ofsubstances, which has a specific binding affinity for the ligand to theexclusion of other substances; and specific binding analog of the ligandis any substance, or group of substances, which behaves essentially thesame as the ligand with respect to the binding affinity of the specificbinding partner for the ligand.

The specific binding reagent means may take many different forms. Ingeneral, such means comprises three basic constituents, which are (1)the ligand to be detected, (2) a specific binding partner of the ligand,and (3) a labeled constituent which is normally a labeled form of (a)the ligand, (b) a specific binding analog of the ligand, or (c) thespecific binding partner. The binding reaction constituents are combinedsimultaneously or in a series of additions, and with an appropriateincubation period or periods the labeled constituent becomes bound toits corresponding competing binding partners such that the extent ofbinding, i.e. the ratio of the amount of labeled constituent bound to abinding partner to that unbound, is a function of the amount of ligandpresent. To follow is a brief description of some of the differentbinding reaction schemes that may be used in carrying out the method ofthe present invention.

While in conventional heterogeneous specific binding assay methods, suchas radioimmunoassays and heterogeneous enzyme immunoassays, the labelingcharacteristic in the labeled conjugate, such as radioactivity orenzymatic activity, is essentially the same for the bound- andfree-forms of the conjugate, according to the present method, theactivity of the reactant, as labeling substance, is in certain casesaffected by binding of the labeled conjugate. In such a situation themonitoring reaction exhibits a relatively constant character where theligand is absent from the liquid medium, or is present in aninsignificantly small amount. When the ligand is present in the liquidmedium, a characteristic or property of the monitoring reaction would bealtered. Generally, the activity of the conjugated reactant would be theextent or rate at which the reactant is capable of participating in themonitoring reaction. Thus, the character of the monitoring reactionwould be altered by the presence of the ligand in the liquid medium,usually with respect to either the aggregate reaction rate thereof orthe equilibrium quantity of one or more reaction products producedthereby. Usually, in this situation, the ability of the conjugatedreactant to participate in the monitoring reaction is decreased uponreaction between the specific binding substance to which it isconjugated and a specific binding counterpart of such specific bindingsubstance, that is, the conjugate in its free state is more active inthe monitoring reaction than in its bound state.

For the diagrams which are set out hereinafter, the following legendshall apply:

    ______________________________________                                        Symbol           Definition                                                   ______________________________________                                        L             ligand to be detected                                           L ○    ligand or specific binding                                                    analog thereof                                                  B             binding partner for the ligand                                  *             labeling substance, i.e.                                                      reactant                                                         ##STR1##      insoluble phase                                                 ##STR2##     incubation period followed by appropriate separation            (lim)         limited; present in an amount                                                 less than that capable of                                                     being bound to the total available                                            binding sites under the                                                       selected reaction conditions                                                  during the selected                                                           incubation period; i.e. the                                                   constituent for which the                                                     other constituents compete for                                                binding                                                         (exc)         excess, present in an amount                                                  greater than that capable of                                                  being bound by the total avail-                                               able binding sites under the                                                  selected reaction conditions                                                  during the selected incubation                                                period                                                          ______________________________________                                    

HETEROGENEOUS ASSAY SCHEMES 1. Competitive binding formats

(a) L+ L *+B(lim)→+insolubilizing agent for B or L *

This is the classical competitive binding approach. Examples of suchinsolubilizing agents are specific precipitating antibodies, specificinsolubilized antibodies, and, where B or L * is a proteinaceousmaterial, protein precipitators such as ammonium sulfate, or where B orL * is a small adsorbable molecule, dextran-coated charcoal. Descriptionof parallel systems may be found in Biochem. J. 88:137(1963) and U.S.Pat. No. 3,839,153.

(b) L+ L *+ B(lim)→

This approach is commonly referred to as the solid-phase technique.Descriptions of parallel radioimmunoassay and enzyme immunoassaytechniques may be found in U.S. Pat. Nos. 3,505,019; 3,555,143;3,646,346; and 3,654,090.

(c) L+B*+ L (lim)→

Reference: U.S. Pat. No. 3,654,090.

(d) L+ L +B*(lim)→

Reference: U.S. Pat. No. 3,850,752.

2. Sequential saturation formats

(a) L+B(exc)→+ L *(exc)→+insolubilizing agent for B or L *

In the sequential saturation technique, some or all the binding sites onB remaining after the first incubation period are bound by the labeledconstituent.

(b) L+ B(exc)→+ L *(exc)→

Descriptions of parallel radioimmunoassay and enzyme immunoassaytechniques may be found in U.S. Pat. No. 3,720,760 and J. Immunol.209:129(1972).

(c) L+B*(exc)→+ L (exc)→

3. "Sandwich" format

L+ B(exc)→B*(exc)→

In the sandwich technique, some or all of the ligand molecules bound tothe insolubilized binding partners are bound by the labeled constituent.

Reference: U.S. Pat. No. 3,720,760.

4. Solid-phase dilution format

L+ L *+ (nonspecific)→+B(lim)→

In this technique, the ligand and the labeled constituent are bound to anon-specific binder and thereafter proportional amounts are dissociatedtherefrom by binding with a binding partner having a greater affinityfor the ligand and the labeled constituent. The most useful form of thistechnique employs a column of the non-specific binder as described inU.S. Pat. No. 3,659,104. Such a technique is useful where the ligand isbound to endogenous binding substances in the sample which unlessremoved would interfer with the competitive binding reaction. Upon beingbound to the non-specific binder, the endogenous binding substances maybe removed by appropriate washes.

For further discussion of the parameters involved in conventionalheterogeneous assay systems, such as more detailed descriptions of assayformats and alternative separation techniques, reference may be had toPrinciples of Competitive Protein-Binding Assays, ed. Odell andDaughaday (J. B. Lippincott Co., Philadelphia, 1972).

It is contemplated that manipulative schemes involving other orders ofaddition and other binding reaction formats may be devised for carryingout heterogeneous specific binding assays without departing from theinventive concept embodied herein.

The step of assessing the activity of the conjugated reactant as aconstituent of the predetermined monitoring reaction system in either ofthe bound- or free-phases is conveniently accomplished by contactingsuch phase with at least one substance which forms with the conjugatedreactant, the monitoring reaction, and measuring a characteristic ofsuch reaction. The monitoring reaction system may comprise a singlechemical transformation or a plurality of series of chemicaltransformations.

Where an enzyme-catalyzed reaction system is used, it includes, inaddition to the conjugated reactant, at least one enzyme and may includeone or more enzymatic reactants such as substrates and coenzymes. Suchenzyme-catalyzed reaction system may comprise a single simple enzymaticreaction or a complex series of enzymatic and non-enzymatic reactions.For instance, the enzyme-catalyzed reaction system may consist of asingle enzyme-catalyzed degradation or dissociation reaction. In such asystem, the conjugated reactant is the enzyme substrate which undergoesdegradation or dissociation, and the only component of the reactionsystem necessary to be contacted with the selected bound- or free-phaseis an enzyme which catalyzes the degradation or dissociation reaction. Amore complex enzyme-catalyzed reaction system may consist of a singleenzymatic reaction involving two or more reactants or may consist of aseries of reactions involving several reactants, at least one of whichreactions is enzyme-catalyzed. In such a system, the conjugated reactantwould be one of the enzymatic reactants in the enzyme-catalyzed reactionand the selected bound- or free-phase would be contacted with theappropriate enzyme and reactant constituents, other than that in theconjugate, necessary to provide the selected enzyme-catalyzed reactionsystem.

It is further contemplated that the enzyme-catalyzed reaction system maycomprise a biochemical system as complex as the metabolic system of abiological cell such as a microorganism. For example, a nutrientsubstance essential to the growth of a particular microorganism may beselected as the reactant in the conjugate. Reactant activity would bemeasurable by monitoring a characteristic of the microorganism, such asthe rate of microorganism growth, when such microorganism would beplaced in an environment wherein the only source of the reactantnutrient substance is the conjugate.

The appropriate reaction constituents which form, together with thereactant in the conjugate, the monitoring reaction system may becontacted with the selected separated phase mixture singularly or in anycombination either prior to, simultaneous with, or subsequent toinitiation of the specific binding reaction. After initiation of thespecific binding reaction, the reaction mixture, which may include anyor all of the necessary components for the monitoring reaction isusually incubated for a predetermined period or periods of time beforeseparation of the resulting bound- and free-phases. After separation,any components which are necessary for the monitoring reaction and whichare not already present in sufficient quantities in the selectedseparated phase are added thereto, and reactant activity therein isassessed as an indication of the presence or amount of the ligand in theliquid medium.

When the reaction rate of the monitoring reaction is the characteristicused to assess reactant activity in the selected bound- or free-phase,as is preferred, such rate is usually determined by measuring the rateof disappearance of a reactant or the rate of appearance of a reactionproduct. Such measurement can be accomplished by a wide variety ofmethods including the conventional chromatographic, gravimetric,potentiometric, spectrophotometric, fluorometric, turbidimetric, andvolumetric analysis techniques. Since the present method is primarilydesigned for the detection of low concentrations of ligands, highlysensitive reaction systems have been developed for use in conjunctionwith the novel specific binding reaction system.

One preferred form of the monitoring reaction includes a luminescentreaction system, preferably enzyme-catalyzed, such as a reactionexhibiting the phenomenon of bioluminescence or chemiluminescence. Thereactant in the conjugate may be a reactant in either thelight-producing reaction or a reaction which is preliminary to anenzymatic or non-enzymatic luminescent reaction. The activity of theconjugated reactant can be assessed by following the rate of lightproduction or the total amount, peak intensity, or character of thelight produced. Examples of luminescent reaction systems are given inTable A in which the following abbreviations are used:

                                      TABLE A                                     __________________________________________________________________________    Luminescent Reaction System            Conjugated Reactant                    __________________________________________________________________________    A.                                                                               ##STR3##                            ATP or reduced luciferin               B.                                                                               ##STR4##                            FMNH.sub.2 or long-chain aldehyde        hν + FMN + long-chain acid + H.sub.2 O                                   C.                                                                               ##STR5##                            NADH or FMN                               ##STR6##                                                                     hν + long-chain acid + H.sub.2 O                                         D.                                                                               ##STR7##                            3',5'-adenosine diphosphate or                                                reduced luciferin                        adenosine-3'-phosphate-5'-phosphosulfate + reduced luciferin                   ##STR8##                                                                   E.                                                                               ##STR9##                            luminol                                F.                                                                               ##STR10##                           reduced pyrogallol                     G.                                                                               ##STR11##                           luminol                                H.                                                                               ##STR12##                           reduced pyrogallol                     I.                                                                               ##STR13##                           isoluminol                             J.                                                                               ##STR14##                           isoluminol                             __________________________________________________________________________     *or catalase                                                                  ATP adenosine triphosphate                                                    AMP adenosine monophosphate                                                   NAD nicotinamide adenine dinucleotide                                         NADH reduced nicotinamide adenine dinucleotide                                FMN flavin mononucleotide                                                     FMNH.sub.2 reduced flavin mononucleotide                                      hν electromagnetic radiation, usually in the infrared, visible, or         ultraviolet region                                                       

Further details and discussion concerning luminescent reaction systemswhich may be used in the present method may be found in the followingreferences:

J. Biol. Chem. 236:48(1961).

J. Amer. Chem. Soc. 89:3944(1967).

Cornier et al. Bioluminescence in Progress, ed. Johnson et al.,Princeton University Press (New Jersey, 1966) pp. 363-84.

Kries, P. Purification and Properties of Renilla Luciferase, doctoralthesis University of Georgia (1967).

Am. J. Physiol. 41:454(1961).

Biol. Bull. 51:89(1962).

J. Biol. Chem. 243:4714(1968).

Another type of preferred, sensitive, monitoring reaction involves thephenomenon of fluorescence and is enzyme catalyzed. In such a reactionsystem the reactant in the conjugate is a substrate in an enzymaticreaction which produces a product which has a fluorescent property whichdistinguishes it from the conjugated substrate. A general reactionscheme for such an enzyme-catalyzed reaction system is as follows:##STR15## wherein X is an enzyme-cleavable bond or linking group, suchas an ester or amido group, and Z is a specific binding substance which,depending upon the specific binding reaction technique used, is theligand, a specific binding analog of the ligand, or a specific bindingpartner of the ligand. Specific conjugates which may be used in thistype of reaction system are various enzyme-cleavable modifications andderivatives of fluorescein, umbelliferone, 3-indole, β-naphthol,3-pyridol, resorufin, and so forth. Examples of possible structuralformulas of such derivatives are as follows:

    ______________________________________                                        Derivative Formula                                                            ______________________________________                                        fluorescein                                                                               ##STR16##                                                         umbelliferone                                                                             ##STR17##                                                         3-indole                                                                                  ##STR18##                                                         β-naphthol                                                                           ##STR19##                                                         3-pyridol                                                                                 ##STR20##                                                         resorufin                                                                                 ##STR21##                                                         ______________________________________                                    

wherein R¹ is --OH or --X--Z (as defined above in this paragraph), R² is--X--Z, and R³ is --H or --CH₃.

A reaction system which is particularly preferably for use in assessingthe activity of the conjugated reactant in the selected separated phaseis a cyclic or cycling reaction system. Such a reaction system is one inwhich a product of a first reaction is a reactant in a second reaction,which second reaction has as one of its products a substance that isalso a reactant in the first reaction.

The following diagram illustrates a model of a cyclic reaction system:##STR22## In the above model cyclic reaction system, a small amount ofcycled material, if provided with sufficient amounts of reactants A andB, will generate large amounts of products A and B. Since the rate andamount of product produced by the reactions constituting the cyclicreaction system is highly sensitive to the amount of cycled materialpresent, it is most preferred to use the cycled material as the reactantin the conjugate of the present invention. Examples of cycling reactionsystems contemplated for use in conjunction with the novel specificbinding reaction system of the present invention are given in TablesB,C, and D.

                  TABLE B                                                         ______________________________________                                         ##STR23##                                                                            reactant A              reactant B                                            or                      or                                            reaction                                                                              product B   enzyme      product A                                     ______________________________________                                        1       lactaldehyde                                                                              alcohol de- propanediol                                                       hydrogenase                                               2       α-ketoglutar-                                                                       glutamic de-                                                                              glutamate                                             ate + NH.sub.3                                                                            hydrogenase                                               3       oxaloacetate                                                                              malic dehy- malate                                                            drogenase                                                 4       acetaldehyde                                                                              alcohol de- ethanol                                                           hydrogenase                                               5       α-ketoglutar-                                                                       isocitric   isocitrate                                            ate + CO.sub.2                                                                            dehydrogen-                                                                   ase                                                       6       dihydroxyace-                                                                             α-glycerol                                                                          L-α-glycerol                                    tone phos-  phosphate   phosphate                                             phate       dehydrogen-                                                                   ase                                                       7       pyruvate    lactic dehy-                                                                              lactate                                                           drogenase                                                 8       1,3-diphos- glyceralde- glyceraldehyde                                        phoglycerate                                                                              hyde-3-phos-                                              3-phosphate                                                                                       phate dehy- + phosphate                                                       drogenase                                                 ______________________________________                                         *nicotinamide adenine dinucleotide                                            **reduced NAD                                                            

                  TABLE C                                                         ______________________________________                                         ##STR24##                                                                            reactant A              reactant B                                            or                      or                                            reaction                                                                              product B   enzyme      product A                                     ______________________________________                                        1       6-phospho-  glucose-6   glucose-6                                             gluconate                                                             phosphate                                                                     phosphate                                                                                         dehydrogenase                                             2       oxidized    glutathione reduced glu-                                          glutathione reductase   tathione                                      3       ρ-benzoqui                                                                            quinone     hydroqui-                                             none        reductase   none                                          4       nitrate     nitrate     nitrite                                                           reductase                                                 5       α-ketoglu-                                                                          glutamic    glutamate                                             tarate + NH.sub.3                                                                         dehydrogenase                                             ______________________________________                                         *nicotinamide adenine dinucleotide phosphate                                  **reduced NADP                                                           

It should be noted that the cyclic reaction systems illustrated inTables B and C comprise the combination of any one of the reactionslisted in the respective tables with any other reaction listed therein.For example, reaction 1 in Table B may be paired with any one ofreactions 2-8 to form a useful cyclic reaction system. Thus, Tables Band C represent respectively 56 and 20 possible cyclic reaction systemsfor use in the present invention.

In addition to the cyclic reaction systems represented in Tables B andC, it is contemplated that one of the reactions in the cyclic reactionsystem may involve the enzymatic or non-enzymatic conversion of aspectrophotometric indicator, preferably colorimetric. An example of acyclic reaction system involving a conversion of an indicator is thesystem produced by combining one of the reactant B--product B reactionsfrom Table B with a reaction comprising an oxidation-reduction indicatorand an electron transfer agent. As electron transfer agent,phenazinemethosulfate may be used. Useful indicators include theoxidized forms of nitrotetrazolium, thiazoyl blue, anddichlorophenolindophenol.

                  TABLE D                                                         ______________________________________                                         ##STR25##                                                                     ##STR26##                                                                     ##STR27##                                                                     ##STR28##                                                                     ##STR29##                                                                     ##STR30##                                                                     ##STR31##                                                                     ##STR32##                                                                     ##STR33##                                                                     ##STR34##                                                                    ______________________________________                                    

In forming any of the cyclic reaction systems illustrated in Tables B,C,and D, where a component in the reaction system is in an ionic form, itmay of course be added in a salt or acid form which is ionizable uponcontacting the liquid medium. A water soluble salt or acid of suchcomponent is usually preferred.

It is also contemplated that an exponential cyclic reaction system maybe included in the monitoring reaction system. An example of anexponential cyclic reaction system is as follows: ##STR35## Such acyclic reaction is autocatalytic in the sense that during each cycle theamount of cycled material is doubled. The cycling rate thereforeincreases exponentially with time and affords a high degree ofsensitivity. Further details and discussion relating to such cyclicreaction may be found in J. Biol. Chem. 247:3558-70(1972).

Where a cyclic reaction system is used as a means of assessing anychange in activity of the conjugated reactant, the rate of disappearanceof a reactant or rate of appearance of a reaction product can bedetermined by conventional techniques or by using one or more additionalcycling systems followed by a conventional determination of theaggregate reaction rate.

The use of a cyclic reaction system in conjunction with theheterogeneous specific binding reaction system provides a high degree ofassay versatility as well as sensitivity. A single reactant-specificbinding substance conjugate may be used with a multiplicity of reactionsto form cyclic systems which have sensitivities varying over a widerange and which provide a wide variety of responses detectable by thesenses or artificial means. Such versatility is lacking in the priorart.

The present invention may be applied to the detection of any ligand forwhich there is a specific binding partner. The ligand usually is apeptide, protein, carbohydrate, glycoprotein, steroid, or other organicmolecule for which a specific binding partner exists in biologicalsystems or can be synthesized. The ligand, in functional terms, isusually selected from the group consisting of antigens and antibodiesthereto; haptens and antibodies thereto; and hormones, vitamins,metabolites and pharmacological agents, and their receptors and bindingsubstances. Specific examples of ligands which may be detected using thepresent invention are hormones such as insulin, chorionic gonadotropin,thyroxine, liothyronine, and estriol; antigens and haptens such asferritin, bradykinnin, prostaglandins, and tumor specific antigens;vitamins such as biotin, vitamin B₁₂, folic acid, vitamin E, andascorbic acid; metabolites such as 3',5' adenosine monophosphate and3',5' guanosine monophosphate; pharmacological agents such as dilantin,digoxin, morphine, digitoxin, and barbiturates; antibodies such asmicrosomal antibody and antibodies to hepatitis and allergens; andspecific binding receptors such as thyroxine binding globulin, avidin,intrinsic factor, and transcobalamine.

In the conjugate of the present invention, the reactant is coupled orbound to a specific binding substance, which is the ligand, a specificbinding analog of the ligand, or a specific binding partner of theligand, depending upon the assay scheme selected, such that a measurableamount of activity of the reactant is retained. The bond between thereactant and the specific binding substance is usually substantiallyirreversible under the conditions of the assay such as where thepredetermined monitoring reaction in which the reactant has activity isnot designed to chemically destroy such bond as in the above-mentionedluminescent and cyclic reaction systems. However, in certain instancessuch bond is by design destroyed or otherwise affected by the selectedmonitoring reaction as a means for assessing in reactant activity. Sucha case is the enzymatic fluorescent substrate reaction systems referredto previously herein.

The reactant may be directly coupled to the specific binding substanceso that the molecular weight of the conjugate is less than or equal tothe aggregate molecular weight of the reactant and the specific bindingsubstance. Usually, however, the reactant and the specific bindingsubstance are linked by a bridge group comprising between 1 and 50, andpreferably between 1 and 10, carbon atoms or heteroatoms such asnitrogen, oxygen, sulfur, phosphorus and so forth. Examples of a bridgegroup comprising a single atom would be a methylene group (one carbonatom) and an amino group (one heteroatom). The bridge group usually hasa molecular weight not exceeding 1000 and preferably less than 200. Thebridge group comprises a chain of carbon atoms or heteroatoms, or acombination of both, and is joined to the reactant and the specificbinding substance, or active derivative thereof, by a connecting groupusually in the form of an ester, amido, ether, thioester, thioether,acetal, methylene, or amino group.

The reactant in the conjugate of the present invention may be anysubstance which has given (i.e. fixed or known) reactant activity as aconstituent of a predetermined monitoring reaction. More particularly,for the purposes of this disclosure, the terms "reactant" and "substancehaving reactant activity" refer to any chemical substance which iscapable of undergoing a finite measurable chemical transformation whichyields one or more products different from itself and which results uponinteraction of said reactant with reaction-initiating means, such as achemical substance (i.e. another reactant, a catalyst, or other typetion), electromagnetic radiation, thermal energy, or sonic energy. Theclass of substances defined herein as "reactants" therefore includesconventional inorganic and organic reagents and various biochemicalmaterials, but excludes such materials as catalysts, including enzymes,and radioactive isotopes which are not reactants in the monitoringreaction. It will be recognized that while a particular chemicalsubstance may be classified in several different catagories because itis able to function in several ways depending on its chemicalenvironment, it is the activity of such substance with respect to theselected monitoring reaction referred to herein which shall govern whichfunctional identity such substance shall have in the context of thisdisclosure.

Preferably, the reactant is an enzymatic reactant such as an enzymesubstrate, a coenzyme, or an active modification or derivative thereof.An enzyme substrate is a compound or moeity capable of undergoing achemical transformation that is catalyzed by an enzyme. Where asubstrate is employed as the conjugated reactant, the preferredmolecular weight thereof is less than 9000 and preferably less than5000. Substrates of such size, because of their lack of molecularcomplexity, are most convenient for use in the fabrication of theconjugate. Examples of enzyme substrates which are contemplated for usein the present invention include the enzyme-cleavable fluorescentsubstrates referred to previously such as fluroescein and umbelliferonederivatives; pH indicators; and spectrophotometric indicator dyes,particularly chromogenic types.

For the above reasons and for reasons of versatility and adaptability,coenzymes are especially preferred for use as the reactant in theconjugate. A coenzyme is a nonprotein molecule which migrates from oneenzyme protein to another in facilitating the efficient performance ofthe catalytic function of the enzyme. All known coenzymes have amolecular weight of less than 9000, the preferred coenzymes having amolecular weight of less than about 5000. Useful coenzymes include thenucleotide coenzymes, particularly those comprising adenine groups, suchas the adenosine phosphates (i.e. the mono-, di-, and tri-phosphateforms), nicotinamide adenine dinucleotide and its reduced forms, andnicotinamide adenine dinucleotide phosphate and its reduced forms. Otheruseful coenzymes include the guanosine phosphates, flavin mononucleotideand its reduced forms, flavin adenine dinucleotide and its reducedforms, coenzyme A and its thioesters including succinyl-coenzyme A,3',5' adenosine diphosphate, andadenosine-3'-phosphate-5'-phosphosulfate.

Useful coenzyme-active conjugates comprise nucleotide coenzymes havingan adenine group to which the specific binding substance, i.e., aligand, a specific binding analog of a ligand, or a specific bindingpartner of a ligand, is coupled through a direct bond or a bridge groupas referred to hereinbefore. Such coenzyme-active conjugates whichcomprise an adenosine phosphate, nicotinamide adenine dinucleotide orits reduced form, or nicotinamide adenine dinucleotide phosphate or itsreduced form, have the following general formula: ##STR36## wherein R¹is ##STR37## wherein R² is ##STR38## wherein R³ is ##STR39## wherein R⁴is ##STR40## wherein R⁵ is --Y--Z; wherein Y is a bond or a bridgegroup; and wherein Z is a ligand, a specific binding analog of a ligand,or a specific binding partner of a ligand. The above formula representsthe ionized forms of the coenzyme-active conjugate, however, theprotonized or acid forms are equally useful. The extent of protonizationdepends on the pH of the environment. Also, the salts of such conjugatesmay also be used where appropriate.

Synthesis of such compounds may be accomplished in a variety of ways. Itis contemplated that the synthesis routes which are schematicallyillustrated below are advantageously followed in the preparation of theuseful compounds. In the illustrated syntheses, the positions on theadenine ring structure are referred to according to the following:##STR41## Also, the following abbreviations are used; ##STR42##

In addition to the compounds mentioned above, useful coenzyme-activeconjugates include the adenosine phosphates to which are coupled thespecific binding substance through the phosphate grouping. Suchcompounds have the following general formula: ##STR43## wherein R² is--Y--Z; wherein Y is a bond or a bridge group; and wherein Z is aligand, a specific binding analog of a ligand, or a specific bindingpartner of a ligand. Also, the protonized or acid forms, as well as thesalt forms where appropriate, may be used.

Synthesis of such compounds may be accomplished in a variety of ways. Itis contemplated that the synthesis routes which are schematicallyillustrated below are advantageously followed in the preparation of theuseful compounds. The abbreviations used hereinbefore also apply to theillustration to follow.

    ______________________________________                                        derivatives of AP                                                             ______________________________________                                        (1)                                                                                ##STR44##                                                                     ##STR45##                                                                     ##STR46##                                                                     ##STR47##                                                                (2)                                                                                ##STR48##                                                                     ##STR49##                                                                     ##STR50##                                                                     ##STR51##                                                                (3)                                                                                ##STR52##                                                                     ##STR53##                                                                     ##STR54##                                                                ______________________________________                                         .sup.(36) Trayer, I.P., et al., Biochem. J. 139:609 (1974).                   .sup.(37) Trayer, I.P., et al., supra.                                   

In one form of the present invention, the components of the specificbinding reaction which are to be combined with the liquid mediumsuspected of containing the ligand are in a liquid or solid form. Theassay method may be carried out in a standard laboratory vessel such asa test tube with the specific binding reaction components and thecomponents of the reaction system being added thereto in solid or liquidform.

It is also contemplated that one or more of the specific bindingreaction components and/or one or more of the components of themonitoring reaction may be incorporated with a carrier. In one aspect,the carrier may be a liquid holding vessel such as a test tube orcapsule containing such component or components in an interior portionthereof, for instance, in the form of a liquid or loose solid or acoating on an interior surface of the vessel. In another aspect, thecarrier may be in the form of matrix which is insoluble and porous, andpreferably absorbent, relative to the liquid medium to be tested. Suchmatrix may be in the form of bibulous papers; polymeric films,membranes, fleeces, or blocks; gels; and so forth. In such a form, thedevice would provide a convenient means for contacting the liquid mediumto be tested, for carrying out the specific binding reaction and/or themonitoring reaction, for effecting the necessary separation, and forobserving the resulting response.

The liquid medium to be tested may be a naturally occurring orartificially formed liquid suspected of containing or known to containthe ligand, and usually is a biological fluid or a liquid resulting froma dilution or other treatment thereof. Biological fluids which may beassayed following the present method include serum, plasma, urine, andamniotic, cerebral, and spinal fluids. Other materials such as solidmatter, for example tissue, or gases may be assayed by reducing them toa liquid form such as by dissolution of the solid or gas in a liquid orby liquid extraction of the solid.

In contrast to the prior art assay systems, biological fluids containingsubstances which have reactant activity similar or identical to that ofthe conjugated labeling substance may be assayed for the ligand withoutbackground interference. Endogenous background reactant activity can bereadily eliminated in several manners. The biological fluid can betreated to selectively destroy the endogenous reactant activity. Suchtreatment may involve the action of a clearing agent which chemicallydestroys the endogenous activity followed by treatment to inactivate thedestructing action of such clearing agent.

For instance, reactant-degrading enzymes often appear naturally inbiological fluids, particularly if the reactant is a coenzyme such asNAD, NADP, or ATP. There are many inhibitors of such coenzyme-degradingenzymes, for example of chelating agents which operate to deprive theenzymes of essential metal ion activators. As a specific example, NADdegrading enzymes are found in normal serum and have sufficientenzymatic activity to remove essentially all endogenous NAD activityfrom isolated serum within a few hours. The degrading activity of suchenzymes may be effectively inhibited by addition of a chelating agentsuch as ethylenediamine tetraacetic acid. Elimination of the degradingactivity may also be accomplished by adding a specific enzyme inhibitor.For example, ATP-degrading enzymes may be inhibited by addition of βγmethylene ATP or αβ methylene ATP.

The present invention will now be illustrated, but is not intended to belimited, by the following Examples.

EXAMPLE 1 Preparation of nicotinamide 6-(2-aminoethylamino) purinedinucleotide

Two (2) grams of nicotinamide adenine dinucleotide (NAD) were dissolvedin 10 ml of water and 0.6 ml of ethyleneimine was added dropwise, the pHbeing maintained below 7 by the addition of 1 M perchloric acid. Whenaddition of ethyleneimine was complete, the pH was adjusted to 4.5 andthe reaction was incubated at 20°-25° C. At 24 hour intervals 0.6 ml ofethyleneimine was added and the pH readjusted to 4.5. After 96 hours,the solution was poured into 10 volumes of acetone at -10° C. The oilwhich formed was collected, washed with ether, and dissolved inapproximately 50 ml of water in a flask.

The resulting solution was adjusted to pH 7.0-7.5 with 1 N sodiumhydroxide, and 1 gram of sodium bicarbonate was added. Nitrogen wasbubbled through the solution for from 4 to 5 minutes and 1 gram ofsodium hydrosulfite was added. The flask was sealed tightly and allowedto stand at room temperature for 45 minutes. The solution was thenoxygenated for 15 minutes and adjusted to pH 11.3 with sodium hydroxide.The solution was heated at 75° C. for 1 hour. Then the reaction mixturewas cooled to room temperature and 0.6 grams oftris-(hydroxymethyl)-aminomethane was added, followed by 5 Nhydrochloric acid to adjust the pH to 7.5. To the resulting solution wasadded 1000 International units of alcohol dehydrogenase and 1 ml ofacetaldehyde. The decreasing optical density of the reaction mixture wasmonitored at 340 nm and when no further decrease was observed, the pHwas adjusted to 3.5. The solution was poured into 10 volumes of acetoneat -10° C. The oil which formed was separated and washed with ether,after which it was dissolved in 10 to 15 ml of water.

The resulting solution was introduced into a 2.5×90 cm column ofSephadex G-10, available from Pharmacia AB, Uppsala, Sweden,equilabrated with water. Fractions of 12 ml volume were collected. Thewavelength of maximum optical absorption in the ultraviolet region andthe optical density at such wavelength were determined for eachfraction. Also, the optical density at 340 nm of each fraction afterreduction with alcohol dehydrogenase was determined. The fractions whichhad an optical absorption maximum at 264 nm and had a ratio of opticaldensity at 340 nm to that at 265 nm greater than 0.05 were pooled. Thepooled material was concentrated to from 15 to 20 ml on a rotaryevaporator and passed through a 2.5×28 cm column of Dowex 1-X8,available from Bio-Rad Laboratories, Richmond, Calif., equilabrated withwater. Additional water was added to wash the pooled material throughthe column, and 10 ml fractions were collected. The fractions which hadan optical density at 340 nm to that at 264 nm greater than 0.1 werepooled.

The pooled material was passed through a 5×45 cm column of Dowex 50-X2,available from Bio-Rad Laboratories, Richmond, Calif., equilibrated withwater. Additional water was added to wash the pooled material throughthe column and 20 ml fractions were collected. The fractions which hadan optical absorption maximum at 264 nm and had a ratio of opticaldensity at 340 nm to that at 265 nm greater than 0.18 were pooled. Thepooled material was concentrated to from 4 to 5 ml and purified byelectrophoresis as follows.

The concentrated material was applied to a sheet of Whatman 3 MM paper,available from Reeve Angel, Clifton, N.J., in a 1 to 2 cm wide stripperpendicular to the direction of current flow. The paper was thenwetted with 0.02 M sodium phosphate at pH 6.0. Electrophoresis wasconducted according to the Durrum hanging paper method, as described inScience 121:829(1955), for 4-7 hours with a potential gradient of about8.5 volts/cm. The location of the desired pyridine nucleotide derivativewas determined by fluorescence developed after spraying a test strip ofthe paper with 0.5 M sodium cyanide according to the procedure describedin J. Biol. Chem. 191:447(1951). The area containing the desiredderivative was cut out of the paper and extracted with three (3) 50 mlvolumes of water. The resulting extracts containing nicotinamide6-(2-aminoethylamine) purine dinucleotide were pooled, concentrated tofrom 3 to 4 ml, and stored at -20° C.

EXAMPLE 2 Preparation of nicotinamide adenine dinucleotide -biotinconjugate

A 16 mg quantity of biotin was suspended in 1 ml of water containing 22mg of nicotinamide 6-(2-aminoethylamino) purine dinucleotide prepared asin Example 1. A few drops of 0.1 N sodium hydroxide was added to aiddissolution of the biotin. A 240 mg quantity of1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-tolulenesulfonate was added to the resulting solution and brought into solutionby dropwise addition of 0.1 N hydrochloric acid. The reaction mixturewas allowed to incubate at room temperature for 5 hours and was thenpoured into 10 ml of acetone at -10° C. The oil which formed wasseparated, washed twice with from 5 to 10 ml of ether and dissolved infrom 1 to 2 ml of water. The resulting material was purified byelectrophoresis on paper as in Example 1. Two fluorescent bands appearedafter spraying with sodium cyanide, one having migrated toward thecathode and the other toward the anode. The latter band, which containedthe NAD-biotin conjugate, was eluted with water and stored at -20° C.

EXAMPLE 3 Preparation of biotin-umbelliferone conjugate(2-Oxo-2-H-1-benzopyran-7-yl)-5-[cis-hexahydro-2-oxo-1H-thieno-(3,4-d-)-imidazole]valericacid ester.

A solution of 300 mg (1.2 mmol) anhydrous biotin in 20 ml drydimethylformamide was stirred at -10° C. under dry nitrogen gas and 0.17ml (1.2 mmol) dry triethylamine was added. A solution of freshlydistilled ethyl chloroformate (0.141 ml in 3 ml of dry ether) was addeddropwise. After incubation for 30 min with stirring, the resultingprecipitate was filtered under a dry nitrogen atmosphere and cooledimmediately to -10° C. To the filtered residue was added a solution of197 mg (1.2 mmol) anhydrous 7-hydroxycoumarin in 3 ml dry pyridine andstirred for 1 hour at -10° C. followed by 20 hours at 25° C. Thesolvents were evaporated under high vacuum at 40° C. After cooling, theresulting solid was filtered and recrystallized from methanol to yieldthe desired product (melting point=216°-218° C.). Calculated for C₁₉ H₂₀N₂ P₅ S: C,48.75; H,4.19; N,7.21. Found: C,58.4; H,5.12; N,6,86.

EXAMPLE 4

Competitive binding-bioluminescence assay for biotin; effect of varyinglevels of biotin on the peak light intensity produced.

The bioluminescence reaction system used in this Example was based onthe following reactions: ##STR55##

A. Preparation of light-generating solution

A light-generating solution for carrying out reactions (b) and (c) wasprepared as follows. A reagent mixture was prepared containing 0.13 Mphosphate buffer at pH 7.0, 0.67 wt% bovine serum albumin, 15.7 μmflavin mononucleotide (FMN), and 13.3 mM sodium acetate, and thismixture was stored in the dark at -20° C. An emulsion of 5 μl ofdodecanal in 5 ml of water was prepared the day the light-generatingsolution was to be used. Lyophilized luciferase extracted fromPhotobacterium fisheri (enzyme available from Worthington BiochemicalCorp., Freehold, N.J.) was added to 0.013 M phosphate buffer at pH 7.3to a concentration of 20 mg/ml. After 30 minutes the resultingsuspension was centrifuged at 1500 xg for 10 minutes and the pellet wasdiscarded. The light -generating solution was then prepared within 5minutes of use by combining 75 μl of the reagent mixture, 5 μl of thedodecanal emulsion, and 20 μl of the luciferase solution.

B. Preparation of insolubilized binding partner.

Avidin, which has a binding affinity for biotin, was insolubilized bybeing covalently bound to a water insoluble polymer bead as follows. Aquantity of Sepharose 4B (available from Pharmacia AB, Uppsala, Sweden)was activated for bonding to avidin using the method of March et al.,Analytical Biochemistry 60:149(1974). Approximately 4 ml of theactivated Sepharose 4B was suspended in 8 ml of 0.1 M citrate buffer atpH 7.0. To the suspension was added 6 mg of avidin, having an activityof 9.9 units/mg, in 3 ml of water. One unit of avidin activity is thatquantity of avidin capable of binding 1 μg of biotin. The resultingreaction mixture was stirred for 6 hours at 7° C. Theavidin-bound-Sepharose 4B was then filtered, washed with 100 ml of 0.1 Msodium bicarbonate buffer at pH 9.0, and resuspended in 240 ml of 0.1 Mtris-(hydroxymethyl)-aminomethane hydrochloride buffer at pH 8.0.

C. Control experiments

Nine specific binding reaction mixtures were prepared, each having atotal volume of 0.19 ml and each containing 0.1 Mtris-(hydroxymethyl)-aminomethane hydrochloride buffer at pH 8.0, 0.6 Methanol, 0.01 M semicarbazide hydrochloride, and respectively theamounts or concentrations indicated in Table 1 of NAD, NAD-biotinconjugate, avidin-bound Sepharose 4B suspension (prepared according toPart B of this Example), and Sepharose 4B suspension (formed bysuspending 1 ml of packed Sepharose 4B in 60 ml of 0.1 Mtris-(hydroxymethyl)-aminomethane hydrochloride buffer at pH 8.0). Thereaction mixtures were shaken gently for 15 minutes at room temperature.Then, 0.22 International units of alcohol dehydrogenase was added toeach reaction mixture to initiate the reduction reaction. Semicarbazidecombines with the acetaldehyde produced in reaction (a) to form asemicarbazone and thus to drive reaction (a) in the desired direction.

The reaction mixtures were shaken again for 15 minutes at roomtemperature. A 10 μl aliquot of the supernatant from each reactionmixture was then injected into a separate cuvette mounted in a DuPontModel 760 Bioluminenscence Photometer (E. I. duPont de Nemours,Wilmington, Del. containing 100 μl of the previously preparedlight-generating solution which had been pre-incubated for from 2 to 3minutes at 25° C. The results appear in Table 1.

                  TABLE 1                                                         ______________________________________                                                       concen-                                                                       tration of                                                                             avidin-                                                              NAD-     bound                                                      concen-   biotin   Sepharose                                                                             Sepharose                                                                             peak                                  reac-                                                                              tration of                                                                              conjugate                                                                              4B suspen-                                                                            4B suspen-                                                                            light                                 tion NAD (nM)  (nM)     sion (μl)                                                                          sion (μl)                                                                          intensity                             ______________________________________                                        1    --        --       --      --      1.2                                   2    21        --       --      --      159                                   3    21        --       20      --      147                                   4    --        10       --      --      45.9                                  5    --        21       --      --      110                                   6    --        21       20      --      28.5                                  7    21        --       --      10      154                                   8    --        21       --      10      114                                   9    --        --       20      --      1.6                                   ______________________________________                                    

The results of control reactions 1 and 9 show that in the absence of NADand NAD-biotin conjugate very little light was produced. Reactions 2 and3 yielded results indicating that the light producing reaction occurredwhen free NAD was added and that such reaction was substantiallyunaffected by the presence of avidin-bound-Sepharose 4B. The results ofreactions 4,5, and 6 show that the NAD-biotin conjugate was active inthe light producing reaction, that the peak light intensity producedincreased as more NAD-biotin conjugate was present, and that thepresence of avidin-bound-Sepharose 4B inhibited light production.Comparison of the results of reactions 3 and 5 with those of 7 and 8shows that the light producing reaction was not affected by the presenceof plain Sepharose 4B.

D. Assay method

Five additional specific binding reaction mixtures were prepared, eachhaving a volume of 0.19 ml and each containing 0.1 Mtris-(hydroxymethyl)-aminomethane hydrochloride buffer at pH 8.0, 0.6 Methanol, 0.01 M semicarbozide, and respectively the amounts orconcentrations indicated in Table 2 of NAD-biotin conjugate, freebiotin, and avidin-bound-Sepharose 4B suspension. Each reaction mixturewas treated in the same manner as the control reaction mixtures in PartC of this Example. The results appear in Table 2.

                  TABLE 2                                                         ______________________________________                                             concentration                                                                             concentration                                                                            avidin-bound                                                                            peak                                    reac-                                                                              of NAD-biotin                                                                             of biotin  Sepharose 4B                                                                            light                                   tion conjugate (nM)                                                                            (nM)       suspension (μl)                                                                      intensity                               ______________________________________                                        10   21          --         --        79.1                                    11   21          --         20        17.4                                    12   21           79        20        43.1                                    13   21          158        20        59.9                                    14   21          158        --        79.7                                    ______________________________________                                    

The results of reactions 11,12, and 13 show that free biotin andNAD-biotin conjugate compete effectively for the binding sites on theinsolubilized avidin since the peak light intensity produced wasdependent upon the amount of free biotin present. Reactions 10 and 14gave results indicating that in the absence of insolubilized avidin, thepeak light intensity produced was constant for vastly differentconcentrations of free biotin.

It was thus demonstrated in this Example that the amount of NAD-biotinconjugate in the liquid phase was inversely related to the amount offree biotin present and thus the assay method and means of the presentinvention are useful in the determination of a ligand in an unknownliquid sample.

EXAMPLE 5

Specific binding assays for avidin and biotin employing an enzymesubstrate as labeling substance.

The specific binding assay systems used in this Example were based onthe following reaction: ##STR56##

A. Preparation of insolubilized binding partner.

Avidin was insolubilized by being covalently bound to a water insolublepolymer bead as in Part B of Example 4 except that after washing with100 ml of 0.1 M sodium bicarbonate buffer at pH 9.0, theavidin-bound-Sepharose 4B was suspended in 12 ml of 0.1 Mtris-(hydroxymethyl) -aminomethane hydrochloride buffer at pH 8.0 anddiluted 1:1 with 0.1 M bis-hydroxyethylglycine hydrochloride buffer atpH 7.0.

B. Competitive binding assay for biotin; effect of various levels ofbiotin on the amount of umbelliferone liberated.

Eight specific binding reaction mixtures were prepared, each having atotal volume of 0.2 ml and each containing 0.1 M bis-hydroxyethylglycinehydrochloride buffer at pH 7.0, 0.3 μM biotin-umbelliferone conjugate(prepared as in Example 3), 15 μl of the avidin-bound-Sepharose 4Bsuspension prepared as in Part A of this Example, and biotin in theconcentrations indicated in Table 3. The reaction mixtures were allowedto incubate at room temperature with gentle shaking for 20 minutes. Eachreaction mixture was centrifuged and a 100 μl aliquot of the supernatantwas combined with 2 ml of 0.1 M bis-hydroxyethylglycine hydrochloridebuffer at pH 8.2 containing 1.08 units of porcine esterase. After a 5minute incubation at room temperature, the fluorescence intensityproduced in each reaction mixture at 448 nm with excitation at 364 nmwas measured using an Amico-Bowman spectrophotofluometer. The resultsappear in Table 3.

                  TABLE 3                                                         ______________________________________                                        reaction   concentration of                                                                             fluorescene                                         mixture    biotin (μM) intensity                                           ______________________________________                                        1          0.00           0.355                                               2          0.10           0.495                                               3          0.20           0.469                                               4          0.30           0.503                                               5          0.40           0.547                                               6          0.50           0.502                                               7          0.75           0.580                                               8          1.00           0.688                                               ______________________________________                                    

It was thus demonstrated in this Example that the amount of NAD-biotinin the liquid phase was directly proportional to the amount of freebiotin present and thus the assay method and means of the present methodare useful in the determination of a ligand in an unknown liquid sample.

What is claimed is:
 1. In a heterogeneous specific binding assay methodfor determining a ligand in a liquid medium, which method comprises thesteps of:(a) contacting said liquid medium with reagent means includinga labeled conjugate comprising a specific binding substance coupled to alabeling substance, said reagent means and the ligand forming a bindingreaction system producing(1) a bound-phase of the labeled conjugate inwhich the specific binding substance therein is bound by a specificbinding partner thereto and (2) a free-phase of the labeled conjugate inwhich the specific binding substance therein is not bound by a specificbinding partner thereto, (b) separating said bound-phase and saidfree-phase; and (c) determining said labeling substance in saidbound-phase or said free-phase as a function of the amount of saidligand in said liquid medium;the improvement wherein said labelingsubstance is a participant in a cyclic chemical reaction of thefollowing type: ##STR57## wherein said labeling substance is a noncatalytic material selected from the group consisting of reactants A,reactants B, and said cycled reactant.
 2. The method of claim 1 whereinsaid labeling substance is determined in said bound-phase or saidfree-phase by forming said cyclic chemical reaction therein andmeasuring the disappearance of one of reactants A or one of reactants B,or the appearance of one of products A or one of products B.
 3. Themethod of claim 1 wherein said labeling substance is said cycledreactant.
 4. The method of claim 3 wherein the reaction of reactants Ato form products A and the reaction of reactants B to form products Bare catalyzed by enzymes and said labeling substance is a coenzyme forsaid enzymes.
 5. The method of claim 4 wherein the same enzyme catalyzesboth the reaction of reactants A to form products A and the reaction ofreactants B to form products B.
 6. The method of claim 5 wherein saidlabeling substance is a coenzyme which cycles between oxidized andreduced forms while participating in said enzyme-catalyzed reactions. 7.The method of claim 6 wherein said labeling substance is flavin adeninedinucleotide.
 8. The method of claim 3 wherein the reaction of reactantsA to form products A and the reaction of reactants B to form products Bare catalyzed by enzymes and said labeling substance is a substrate forsaid enzymes.
 9. The method of claim 1 wherein said cyclic reaction isexponential in that one of products A or products B is also said cycledreactant.
 10. The method of claim 9 wherein said labeling substance issaid cycled reactant.
 11. The method of claim 1 wherein said ligand isselected from the group consisting of antigens and antibodies thereto;haptens and antibodies thereto; and hormones, vitamins, metabolites, andpharmacological agents, and their receptors and binding substances. 12.The method of claim 1 wherein said liquid medium is a biological fluid.13. The method of claim 1 wherein said labeling substance has amolecular weight of less than
 9000. 14. In a reagent means for use in aheterogeneous specific binding assay method for determining a ligand ina liquid medium, which means includes (i) a labeled conjugate comprisinga specific binding substance coupled to a labeling substance having apredetermined characteristic, which means and the ligand form a bindingreaction system producing a bound-phase and a free-phase of the labeledconjugate, one of such phases being insoluble and the other beingsoluble, the amount of said predetermined characteristic of the labelingsubstance in said bound-phase or said free-phase being a function of theamount of the ligand present in the liquid medium under assay, and (ii)a specific binding partner of said ligand which binding partner is in aform which is insoluble in said liquid medium,the improvement whereinsaid labeling substance is a participant in a cyclic chemical reactionof the following type: ##STR58## wherein said labeling substance is anon catalytic material selected from the group consisting of reactantsA, reactants B, and said cycled reactant, wherein the conjugate and theimmobilized specific binding partner are present in amounts sufficientto determine said ligand.
 15. The means of claim 14 wherein saidlabeling substance is said cycled reactant.
 16. The means of claim 15wherein the reaction of reactants A to form products A and the reactionof reactants B to form products B are catalyzed by enzymes and saidlabeling substance is a substrate for said enzymes.
 17. The means ofclaim 16 wherein said labeling substance is α-aminoacid or α-ketoacid,and said means additionally comprises L-aminoacid oxidase, glutamate,and transaminase.
 18. The means of claim 16 wherein said labelingsubstance is oxidized or reduced glutathione, and said meansadditionally comprises nicotinamide adenine dinucleotide phosphate,glutathione reductase, dehydroascorbate, and dehydroascorbate reductase.19. The means of claim 16 wherein said labeling substance is ascorbateor dehydroascorbate, and said means additionally comprises ascorbateoxidase, oxidized glutathione, and dehydroascorbate reductase.
 20. Themeans of claim 16 wherein said labeling substance is oxidized or reducedcytochrome C and said means additionally comprises nicotinamide adeninedinucleotide phosphate, cytochrome C reductase, hydrogen peroxide, andcytochrome C peroxidase.
 21. The means of claim 16 wherein said labelingsubstance is oxidized or reduced ferridoxin, and said means additionallycomprises hydrogen, hydrogenase, nicotinamide adenine dinucleotidephosphate, and pyridine nucleotide reductase.
 22. The means of claim 14wherein the reaction of reactants A to form products A and the reactionof reactants B to form products B are catalyzed by enzymes and saidlabeling substance is a coenzyme for said enzyme.
 23. The means of claim22 wherein said labeling substance is nicotinamide adenine dinucleotideor a reduced form thereof, and, in order to form said cyclic reaction,said means additionally comprises:(1) one of the following groups ofsubstances(a) lactaldehyde and alcohol dehydrogenase; (b)α-ketoglutarate, a substance capable of releasing ammonia upon contactwith said liquid medium, and glutamic dehydrogenase; (c) acetaldehydeand alcohol dehydrogenase; (d) α-ketoglutarate, a substance capable ofreleasing carbon dioxide upon contact with said liquid medium, andisocitric dehydrogenase; (e) dihydroxyacetone phosphate and α-glycerolphosphate dehydrogenase; (f) pyruvate and lactate dehydrogenase; (g)1,3-diphosphoglycerate and glyceraldehyde-3-phosphate dehydrogenase; or(h) oxaloacetate and malic dehydrogenase; and (2) one of the followinggroups of substances other than the group having the same referenceletter as the group selected from groups (1) above(a) propanediol andalcohol dehydrogenase; (b) glutamate and glutamic dehydrogenase; (c)ethanol and alcohol dehydrogenase; (d) isocitrate and isocitricdehydrogenase; (e) L-α-glycerol phosphate and α-glycerol phosphatedehydrogenase; (f) lactate and lactic dehydrogenase; (g)glyceraldehyde-3-phosphate, phosphate, and glyceraldehyde-3-phosphatedehydrogenase; or (h) malate and malic dehydrogenase.
 24. The means ofclaim 22 wherein said labeling substance is nicotinamide adeninedinucleotide phosphate or a reduced form thereof and, in order to formsaid cyclic reaction, said means additionally comprises:(1) one of thefollowing groups of substances(a) oxidized glutathione and glutathionereductase; (b) ρ-benzoquinone and quinone reductase; (c) nitrate andnitrate reductase; (d) 6-phosphogluconate and glucose-6-phosphatedehydrogenase; or (e) α-ketoglutarate, a substance capable of releasingammonia upon contact with said liquid medium, and glutamicdehydrogenase; and (2) one of the following groups of substances otherthan the group having the same reference letter as the group selectedfrom groups (1) above(a) reduced glutathione and glutathione reductase;(b) hydroquinone and quinone reductase; (c) nitrite and nitratereductase; (d) glucose-6-phosphate and glucose-6-phosphatedehydrogenase; or (e) glutamate and glutamic dehydrogenase.
 25. Themeans of claim 22 wherein said labeling substance is flavinmononucleotide or a reduced form thereof, and said means additionallycomprises nicotinamide adenine dinucleotide phosphate, oxidizedcyctochrome C, and cyctochrome C reductase.
 26. The means of claim 22wherein said labeling substance is flavin adenine dinucleotide or areduced form thereof, and said means additionally comprises D-aminoacidand D-aminoacid oxidase.
 27. The means of claim 22 wherein said labelingsubstance is adenosine diphosphate or adenosine triphosphate, and saidmeans additionally comprises phosphoenol pyruvate, pyruvate kinase, andadenosine triphosphatase.
 28. The means of claim 22 wherein saidlabeling substance is coenzyme A or succinyl-coenzyme A, and said meansadditionally comprises α-ketoglutarate, nicotinamide adeninedinucleotide, α-ketoglutarate dehydrogenase, phosphate, guanosinediphosphate, and succinic thiokinase.
 29. The means of claim 22 whereinsaid labeling substance is guanosine triphosphate or guanosinediphosphate, and said means additionally comprises oxaloacetate,phosphoenol pyruvate kinase, adenosine triphosphate, and nucleosidediphosphate kinase.
 30. The means of claim 22 wherein said cyclicreaction is exponential in that one of products A or products B is alsosaid cycled reactant.
 31. The means of claim 30 wherein said labelingsubstance is adenosine triphosphate or adenosine diphosphate, and saidmeans additionally comprises adenosine monophosphate, myokinase,phosphoenolpyruvate, and pyruvate kinase.
 32. The means of claim 22wherein the same enzyme catalyzes both the reaction of reactants A toform products A and the reaction of reactants B to form products B. 33.The means of claim 32 wherein said labeling substance is a coenzymewhich cycles between oxidized and reduced forms while participating insaid enzyme-catalyzed reactions.
 34. The means of claim 33 wherein saidlabeling substance is flavin adenine dinucleotide.
 35. The means ofclaim 14 wherein said cyclic reaction is exponential in that one ofproducts A or products B is said cycled reactant.
 36. The means of claim35 wherein said labeling substance is said cycled reactant.
 37. Themeans of claim 14 wherein said ligand is selected from the groupconsisting of antigens and antibodies thereto; haptens and antibodiesthereto; and hormones, vitamins, metabolites, and pharmacologicalagents, and their receptors and binding substances.
 38. The means ofclaim 14 wherein at least one of the components of said means isincorporated with a carrier that is insoluble in said liquid medium. 39.The means of claim 38 wherein said carrier is a matrix that is absorbentrelative to said liquid medium.
 40. The means of claim 14 wherein saidlabeling substance has a molecular weight of less than
 9000. 41. Themeans of claim 14 which comprises (i) a soluble labeled conjugatecomprising said ligand, a specific binding analog thereof, or a specificbinding partner thereof, coupled to said labeling substance, and (ii) aninsoluble specific binding partner of said ligand.